A Novel Ligand-binding Site in the ζ-Form 14-3-3 Protein Recognizing the Platelet Glycoprotein Ibα and Distinct from the c-Raf-binding Site
1998; Elsevier BV; Volume: 273; Issue: 50 Linguagem: Inglês
10.1074/jbc.273.50.33465
ISSN1083-351X
Autores Tópico(s)HER2/EGFR in Cancer Research
ResumoWe reported previously that the ζ-form 14-3-3 protein (14-3-3ζ) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbα. In this study, we have identified a binding site in the helix I region of 14-3-3ζ (residues 202–231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbα. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3ζ requires helix G (residues 163–187) but not helix I. Thus, the GPIbα-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3ζ has a higher affinity for GPIb-IX complex than recombinant GPIbα cytoplasmic domain. Deletion of helices A and B (residues 1–32) disrupts 14-3-3ζ dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3ζ dimerization, however, does not reduce 14-3-3ζ binding to recombinant GPIbα cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3ζ dimer interacts with both GPIbα and GPIbβ (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding. We reported previously that the ζ-form 14-3-3 protein (14-3-3ζ) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbα. In this study, we have identified a binding site in the helix I region of 14-3-3ζ (residues 202–231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbα. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3ζ requires helix G (residues 163–187) but not helix I. Thus, the GPIbα-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3ζ has a higher affinity for GPIb-IX complex than recombinant GPIbα cytoplasmic domain. Deletion of helices A and B (residues 1–32) disrupts 14-3-3ζ dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3ζ dimerization, however, does not reduce 14-3-3ζ binding to recombinant GPIbα cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3ζ dimer interacts with both GPIbα and GPIbβ (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding. A platelet receptor for von Willebrand factor, the glycoprotein (GP) 1The abbreviations used are: GP, glycoprotein; GPIb-IX, glycoprotein Ib-IX complex; PKA, protein kinase A; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PCR, polymerase chain reaction; pS, phosphoserine. 1The abbreviations used are: GP, glycoprotein; GPIb-IX, glycoprotein Ib-IX complex; PKA, protein kinase A; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PCR, polymerase chain reaction; pS, phosphoserine. Ib-IX-V complex (GPIb-IX-V), mediates initial platelet adhesion to the subendothelial matrix and triggers platelet activation under high shear rate conditions (for reviews, see Refs. 1Ware J. Thromb. Haemostasis. 1998; 79: 466-478Crossref PubMed Scopus (66) Google Scholar and 2Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar). GPIb-IX-V also binds thrombin and is important in thrombin-induced platelet activation (3Okumura T. Jamieson G.A. Thromb. Res. 1976; 8: 701-706Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 4Greco N.J. Tandon N.N. Jones G.D. Kornhauser R. Jackson B. Yamamoto N. Tanoue K. Jamieson G.A. Biochemistry. 1996; 35: 906-914Crossref PubMed Scopus (68) Google Scholar, 5De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar). GPIb-IX-V consists of four different transmembrane subunits as follows: disulfide-linked GPIbα and GPIbβ forms a 1:1 complex with GPIX (6Du X. Beutler L. Ruan C. Castaldi P.A. Berndt M.C. Blood. 1987; 69: 1524-1527Crossref PubMed Google Scholar); the GPIb-IX complex (GPIb-IX) forms a 2:1 complex with GPV which may dissociate in certain detergents such as Triton X-100 (7Modderman P.W. Admiraal L.G. Sonnenberg A. von dem Borne A. J. Biol. Chem. 1992; 267: 364-369Abstract Full Text PDF PubMed Google Scholar). Accumulating evidence indicates that ligand binding to GPIb-IX-V triggers transmembrane signaling events including activation of protein kinase C (8Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (235) Google Scholar, 9Kroll M.H. Hellums J.D. Guo Z. Durante W. Razdan K. Hrbolich J.K. Schafer A.I. J. Biol. Chem. 1993; 268: 3520-3524Abstract Full Text PDF PubMed Google Scholar) and tyrosine kinases (10Jackson S.P. Schoenwaelder S.M. Yuan Y. Rabinowitz I. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 27093-27099Abstract Full Text PDF PubMed Google Scholar), elevation of intracellular calcium (8Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (235) Google Scholar, 11Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 12Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K. Sakai K. Mayumi F. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar), synthesis of thromboxane A2 (8Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (235) Google Scholar), and activation of phosphoinositol 3-kinase (10Jackson S.P. Schoenwaelder S.M. Yuan Y. Rabinowitz I. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 27093-27099Abstract Full Text PDF PubMed Google Scholar), leading to activation of ligand binding function of the responsive adhesion receptor, integrin αIIbβ3. We found that GPIb-IX is physically associated with an intracellular signaling protein, the ζ-form 14-3-3 protein (14-3-3ζ) (13Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar), suggesting a potential role for 14-3-3ζ in GPIb-IX-V mediated signaling. We further identified a C-terminal 5-residue sequence of GPIbα (SGHSL) that is critical for the binding of 14-3-3ζ (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), a result confirmed by Andrew et al. (15Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar). Interestingly, in addition to the C-terminal sequence of GPIbα, GPIb-IX binding to 14-3-3ζ also involves a protein kinase A (PKA)-phosphorylated binding site in the cytoplasmic domain of GPIbβ (15Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 16Calverley D.C. Kavanagh T.J. Roth G.J. Blood. 1998; 91: 1295-1303Crossref PubMed Google Scholar). 14-3-3ζ belongs to the 14-3-3 family of highly conserved intracellular proteins (17Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). The 14-3-3 proteins are dimeric; each monomer is composed of nine anti-parallel α-helices forming a large ligand binding groove as revealed by crystal structure analysis (18Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (435) Google Scholar,19Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (398) Google Scholar). The 14-3-3 proteins bind and regulate a variety of intracellular signaling molecules, including various protein kinase C isotypes (17Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar,20Meller N. Liu Y.C. Collins T.L. Bonnefoy B.N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar), c-Raf (21Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (241) Google Scholar, 22Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (351) Google Scholar, 23Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 24Fanti W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (307) Google Scholar), Bcr (25Reuther G.W. Fu H. Cripe L.D. Collier R.J. Pendergast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar), middle T antigen (26Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roberts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar), c-Cbl (27Liu Y.C. Elly C. Yoshida H. Bonnefoy-Berard N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), cdc25 (28Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar), and the cell death factor BAD (29Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar). The 14-3-3 proteins also have been implicated in the assembly of protein kinase complexes (30Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar). Thus, it is possible that binding of GPIb-IX to 14-3-3ζ links GPIb-IX to intracellular signaling pathways. In addition, the 14-3-3-binding site in GPIbα has been shown (31Dong J.F. Li C.Q. Sae T.G. Hyun W. Afshar K.V. Lopez J.A. Biochemistry. 1997; 36: 12421-12427Crossref PubMed Scopus (47) Google Scholar) to be important in regulating the lateral movement of GPIb-IX-V in plasma membrane, suggesting its possible involvement in regulating GPIbα interaction with the cytoskeleton. Thus, structural definition of the 14-3-3ζ-GPIb-IX interaction may serve as a basis for understanding the role of 14-3-3 in signaling mediated by GPIb-IX. Many of the 14-3-3-binding proteins contain an Arg-Ser-X-phosphoserine-X-Pro (RSXpSXP) motif, originally found in c-Raf (32Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar). At least two of the RSXpSXP motif-containing ligands c-Raf and tryptophan hydroxylase can be induced to bind 14-3-3 by PKA-catalyzed phosphorylation (32Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, 33Banik U. Wang G.A. Wagner P.D. Kaufman S. J. Biol. Chem. 1997; 272: 26219-26225Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Other serine protein kinases have also been shown to induce 14-3-3 binding to various proteins (27Liu Y.C. Elly C. Yoshida H. Bonnefoy-Berard N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,34Ichimura T. Uchiyama J. Kunihiro O. Ito M. Horigome T. Omata S. Shinkai F. Kaji H. Isobe T. J. Biol. Chem. 1995; 270: 28515-28518Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Recently, several different 14-3-3 binding sequences have been identified in 14-3-3 ligands including RX(Y/F)XpSXP, RXXSXpSXP, and RXSX(pS/pT)XP (where pT is phosphothreonine) (15Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 27Liu Y.C. Elly C. Yoshida H. Bonnefoy-Berard N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 35Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). In the cytoplasmic domain of GPIbα, a 5-residue sequence, SGHSL, is both necessary and sufficient for interaction with 14-3-3ζ (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar). Interestingly, in contrast to RSXpSXP-containing ligands c-Raf and tryptophan hydroxylase, the cytoplasmic domain of GPIbα appears to be a poor PKA substrate in intact platelets (36Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar, 37Fox J.E. Berndt M.C. J. Biol. Chem. 1989; 264: 9520-9526Abstract Full Text PDF PubMed Google Scholar). Thus, characterization of the structural basis of the GPIbα interaction with 14-3-3 will clarify the differences between RSXpSXP-containing 14-3-3 ligands and GPIbα and further our understanding of how 14-3-3 regulates functions of different types of ligand proteins. In this study, we have identified a binding site in 14-3-3ζ in the helix I region of 14-3-3ζ encompassing residues 202–231 that interact with the GPIbα cytoplasmic domain and the intact GPIb-IX complex. We also show in vitro that c-Raf binds to 14-3-3ζ in a PKA-dependent manner and that this binding requires helix G but not helix I. Thus, the GPIbα-binding site in 14-3-3ζ is distinct from the binding site for RSXpSXP-containing ligand c-Raf. Furthermore, we show that 14-3-3ζ dimerization is required for high affinity binding to GPIb-IX complex, suggesting that a dual site recognition mechanism involving GPIbα and β subunits and dimerized 14-3-3ζ. Cloning of the cDNA encoding wild type 14-3-3ζ was described previously (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The 14-3-3ζ cDNA was subcloned into a pmalC2 vector (New England Biolabs, Beverly, MA). The construct (pmal1433ζ) encodes a fusion protein with the N-terminal region corresponding to the Escherichia coli maltose-binding protein (MBP) and C-terminal region corresponding to 14-3-3ζ. Mutagenesis of pmal1433ζ was performed using PCR techniques (38Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar). In all mutants except T3, stop codons were introduced into reverse primers at designated sites of the 14-3-3ζ. The stop codon in mutant T3 was introduced inadvertently by PCR error. The mutants were subcloned into a pmalC2 vector at the EcoRI and XbaI sites. Correct sequences were verified by automated sequencing. The wild type 14-3-3ζ and 14-3-3 truncation mutants were purified by affinity chromatography using a cross-linked amylose-Sepharose column (New England Biolabs, Beverly, MA). Equivalent amounts (71 μmol of protein/ml Sepharose) of the purified wild type or mutant 14-3-3ζ or MBP were conjugated onto cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech), respectively. Coupling efficiencies in all cases were better than 99% as assessed by optical density at 280 nm wave length. The cDNA encoding GPIbα in a pBlueScript vector was a generous gift from Dr. Jerry Ware at the Scripps Research Institute, La Jolla, CA. The cDNA fragment encoding the cytoplasmic domain of GPIbα (residues 518–610) was generated by PCR with EcoRI andXbaI sites incorporated in the forward and reverse primers, respectively. The PCR product was subcloned into the pmalC2 vector. The correct sequence was verified by automated sequencing. The protein was expressed and purified as described previously (38Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar). Specific binding of the platelet GPIb-IX complex to 14-3-3ζ-conjugated beads has been described previously (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Briefly, washed platelets were resuspended in Hepes buffer (137 mmNaCl, 2.7 mm KCl, 1 mm MgCl2, 5.6 mmd-glucose, 3.3 mmNa2HPO4, 3.8 mm Hepes, pH 7.35) and solubilized by adding an equal volume of the solubilization buffer (2% Triton X-100, 0.1 m Tris, 0.01 m EGTA, 0.15m NaCl, and 1 mm dithiothreitol, pH 7.4) containing 0.2 mm E64 (Boehringer Mannheim) and 1 mm phenylmethylsulfonyl fluoride (13Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar). In some experiments, platelets were solubilized in the presence of 1 mmCaCl2 but in the absence of EGTA and E64 to allow calpain cleavage of GPIbα and thus generation of the C-terminal domain of GPIb-IX complex. After centrifugation at 100,000 × gfor 30 min, the lysates (200 μl) were incubated with 25 μl (50% (v/v)) MBP-conjugated control beads or 14-3-3ζ-conjugated beads at 4 °C for 1 h. The beads were then washed three times in a 1:1 mix of Hepes buffer and solubilization buffer. Bound proteins were extracted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Western blotting with a monoclonal antibody against GPIbα, WM23 (kindly provided by Dr. Michael C. Berndt, Baker Institute, Melbourne, Australia (39Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (150) Google Scholar)). In some experiments, GPIb-IX was also detected using a polyclonal anti-peptide antibody against the C-terminal domain of GPIbα, anti-IbαC (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Reactions with antibodies were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech), and Kodak X-Omat AR film. In some experiments, reactions were visualized using SuperSignal chemiluminescence substrate (Pierce) and quantitated with a Bio-Rad PhosphorImager and chemiluminescence-sensitive phosphor-screens that have a theoretical linear range of 100–104. Binding of recombinant GPIbα cytoplasmic domain fusion protein (Mal-IbαC) to 14-3-3ζ was performed essentially as described above except purified Mal-IbαC fusion protein was used. The Mal-IbαC binding was performed in the platelet lysate buffer described above or in 0.1m sodium citrate, pH 5.6. Binding of Mal-IbαC protein to beads was detected by Western blotting with a rabbit anti-peptide antibody against the cytoplasmic domain of GPIbα, anti-IbαC (14Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Human recombinant c-Raf cDNA (a gift from Dr. Michael Karin, University of California at San Diego, La Jolla) was subcloned into a pmalC2 vector by three-fragment ligation (EcoRI, HindIII, andXbaI) and expressed as a fusion protein with maltose-binding protein. The purified recombinant c-Raf was phosphorylated by incubation with protein kinase A catalytic subunit (10 units/100 μl) (Sigma) and 1 mm ATP in 15 mm Hepes, 5 mm magnesium acetate, 0.1 mm EGTA, 130 mm KCl, 1 mg/ml bovine serum albumin, 1 mmdithiothreitol, pH 7.4, at 22 °C for 30 min. The PKA-treated c-Raf was incubated with 14-3-3ζ-conjugated beads at 4 °C for 1 h. After three washes, bead-bound proteins were then analyzed by SDS-PAGE and immunoblotted with an antibody against c-Raf (Santa Cruz Biotechnology). Purified MBP-14-3-3ζ fusion protein and 14-3-3 mutants (200 μl, ∼1.5 mg/ml) were analyzed by gel filtration using a Pharmacia Superdex 200 HR 10/30 column and a Pharmacia Explorer 10 high performance liquid chromatography system at a flow rate of 0.5 ml/min. The column was equilibrated with 0.02 m Tris, 0.15m NaCl, pH 7.4. The molecular mass was determined by comparing with the elution volumes of the molecular mass standard proteins, IgG (150 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa). In order to identify the sequence within 14-3-3ζ that is responsible for its interaction with the GPIb-IX complex, we made various 14-3-3ζ truncation mutants (Fig.1). Mutants T1 to T6 were truncated from the C-terminal end of 14-3-3ζ. Mutants T7–T9 were truncated progressively from the N-terminal end of the protein. T11-(136–209) encompasses helices G and H, and T12 contains helices H and I. T13-(188–209) contains a single helix H that was implicated in tryptophan hydroxylase binding (34Ichimura T. Uchiyama J. Kunihiro O. Ito M. Horigome T. Omata S. Shinkai F. Kaji H. Isobe T. J. Biol. Chem. 1995; 270: 28515-28518Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). T14-(202–231) containing helix I was generated when results obtained with T1–T13 indicated the location of the GPIbα-binding site (see below). As 14-3-3ζ is composed of nine anti-parallel α-helices (A-I), truncation sites in all these mutants are located between two neighboring α-helices as determined by the published crystal structure to avoid disruption of each of these helical structures (Fig. 1). These mutant proteins were expressed as fusion proteins with maltose-binding protein. Equivalent amounts of the purified wild type and mutant 14-3-3ζ were conjugated to cyanogen bromide-activated Sepharose 4B as described previously (38Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar). We first examined the binding of Triton X-100-solubilized platelet GPIb-IX to the above described Sepharose beads conjugated with 14-3-3 truncation mutants (T1–T13). MBP-conjugated beads were used as a negative control, and the wild type 14-3-3ζ-beads was used as a positive control. Bead-bound GPIb-IX was detected by Western blotting with an anti-GPIbα antibody, WM23, and quantitated with a Bio-Rad PhosphorImager and chemiluminescence-sensitive phosphor-screens. Fig.2 shows that GPIb-IX binds to wild type 14-3-3ζ- but not MBP-conjugated beads. Removal of the C-terminal tail of 14-3-3ζ by truncation at amino acid residue 231 (mutant T6) did not negatively affect GPIb-IX binding, suggesting that the C-terminal region between residues 231 and 246 is not required. Further truncation at residue 209 (mutant T5), which removes the helix I (the first α-helix from the C-terminal end), completely abolished GPIb-IX binding (Fig. 2). Furthermore, none of the truncation mutants that lack helix I bound to GPIb-IX (T5, T11, T13, Fig. 2; T1-T4, data not shown). Thus, the helix I region encompassing residues 209–231 of 14-3-3ζ appears to be required for binding to GPIb-IX. Truncation mutants T9-(188–246) and T12-(188–231) containing helix H and I interacted with GPIb-IX, indicating that the sequence between residues 188 and 231 of 14-3-3ζ (helices H and I) contains a binding site for the GPIb-IX complex. Similar results were also obtained when bead-bound GPIb-IX was detected with an antibody against the C-terminal region of GPIbα, anti-IbαC (data not shown). Fig. 2 also shows that truncation mutants of 14-3-3ζ lacking the N-terminal domain (T7, T8, T9, and T12) binds significantly less GPIb-IX in comparison with wild type 14-3-3ζ. Since similar amounts of proteins were conjugated to these beads (Fig. 2 C), this result indicates that GPIb-IX bound to these mutants with reduced affinity. In particular, the mutant T7 lacking only 33 residues in helices A and B (1Ware J. Thromb. Haemostasis. 1998; 79: 466-478Crossref PubMed Scopus (66) Google Scholar, 2Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 3Okumura T. Jamieson G.A. Thromb. Res. 1976; 8: 701-706Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 4Greco N.J. Tandon N.N. Jones G.D. Kornhauser R. Jackson B. Yamamoto N. Tanoue K. Jamieson G.A. Biochemistry. 1996; 35: 906-914Crossref PubMed Scopus (68) Google Scholar, 5De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 6Du X. Beutler L. Ruan C. Castaldi P.A. Berndt M.C. Blood. 1987; 69: 1524-1527Crossref PubMed Google Scholar, 7Modderman P.W. Admiraal L.G. 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Previously reported crystal structural analysis of 14-3-3ζ has revealed that the N-terminal helices (A and B) are involved in the formation of 14-3-3ζ dimers (18Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (435) Google Scholar, 19Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (398) Google Scholar). Thus, it is possible that the reduced affinity for GPIb-IX is caused by disruption of dimerization. To examine this possibility, we determined the molecular mass of the MBP-14-3-3ζ fusion proteins by gel filtration chromatography under non-denaturing conditions. The 14-3-3ζ monomer has a molecular mass of ∼30 kDa (40Zupan L.A. Steffens D.L. Berry C.A. Landt M. Gross R.W. J. Biol. Chem. 1992; 267: 8707-8710Abstract Full Text PDF PubMed Google Scholar). MBP has a m
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